Nanopore metagenomic sequencing of full length human metapneumovirus (HMPV) within a unique sub-lineage

Human metapneumovirus (HMPV) has been recognized as an important pathogen which can cause a spectrum of respiratory tract disease. Here, we report Nanopore metagenomic sequencing of the first full length HMPV genome directly from a throat swab from a UK patient with complex lung disease and immunocompromise. We found a predominance (26.4%) of HMPV reads in the metagenomic sequencing data and consequently assembled the full genome at a high depth of coverage (mean 4,786). Through phylogenetic analyses, we identified this HMPV strain to originate from a unique genetic group in A2b, showing the presence of this group in the UK. Our study demonstrated the effectiveness of Nanopore metagenomic sequencing for diagnosing infectious diseases and recovering complete sequences for genomic characterization, highlighting the applicability of Nanopore sequencing in clinical settings. Importance Nanopore metagenomic sequencing has the potential to evolve as a point-of-care test for a range of infectious diseases. Here, we report the first full length human metapneumovirus (HMPV) genome in the UK sequenced by Nanopore from a non-invasive sample from an immunocompromised patient. We demonstrate the presence of HMPV from a unique genetic group not previously reported from the UK. Our study demonstrates the effectiveness of Nanopore sequencing for diagnosing an infection that was not detected by routine first-line tests in the clinical microbiology laboratory. We report sufficient genomic data to provide insight into the epidemiology of infection and with the potential to inform treatment decisions.

the full genome at a high depth of coverage (mean 4,786). Through phylogenetic analyses, Nanopore metagenomic sequencing has the potential to evolve as a point-of-care test for a 31 range of infectious diseases. Here, we report the first full length human metapneumovirus 32 (HMPV) genome in the UK sequenced by Nanopore from a non-invasive sample from an 33 immunocompromised patient. We demonstrate the presence of HMPV from a unique genetic 34 group not previously reported from the UK. Our study demonstrates the effectiveness of 35 Nanopore sequencing for diagnosing an infection that was not detected by routine first-line 36 tests in the clinical microbiology laboratory. We report sufficient genomic data to provide 37 insight into the epidemiology of infection and with the potential to inform treatment 38 decisions. A2b, B1, and B2) have been described [4].

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The Nanopore sequencing platform (Oxford Nanopore Technology, ONT) is capable of 54 generating real-time sequencing data, with the potential to evolve as a point-of-care test for a 55 range of infectious diseases [5,6]. In this report, we describe recovery of full length HMPV 56 genome directly from a throat swab through the application of Nanopore metagenomic 57 sequencing.

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A male in his 40's with cystic fibrosis (CF) and a previous lung transplant presented with 59 breathlessness, thick sputum and low oxygen saturations. His condition was further 60 complicated by CF-related diabetes mellitus and bronchiolitis obliterans. To our knowledge, 61 he had not travelled outside the UK. As he presented to hospital during the peak of the 62 influenza season, a throat swab was taken to test for respiratory viruses in a clinical 63 diagnostic laboratory; this sample was negative by PCR for influenza A, influenza B, and 64 respiratory syncytial virus. Given his previous confirmed colonisation with Pseudomonas 65 aeruginosa, he was treated with broad spectrum intravenous antibiotics, and discharged from 66 hospital after two weeks. 67 We performed Nanopore metagenomic sequencing and generated 168,811 reads from this 68 throat swab. We identified 44,580 (26.4%) HMPV reads and 5,393 (3.1%) human reads 69 (which were discarded and not retained). The remaining reads mostly comprised bacteria at a high mean depth of coverage (4,786). The mean alignment length was 1,534bp and 25% 74 of the alignments were longer than 2,000bp ( Fig. 1). We used an alignment-based approach to recover a HMPV genomic sequence of 12,893bp, referred to as JR001 (accession number 76 xxx). The sequence is nearly complete excepting 205bp at the start of the coding region.

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To determine the relationship between JR001 and previously published HMPV genomes,  The extent to which the virus is a pathogen in this context is uncertain, as the patient was  (Fig. S1).

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The case is the first full length HMPV genome in the UK sequenced by Nanopore

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Sample collection, preparation, and Nanopore sequencing 117 A throat swab was collected in viral transport media from a patient presenting to our 118 tertiary referral teaching hospital in Oxford, United Kingdom. The sample was tested for 119 respiratory viruses using Xpert Xpress Flu/RSV assay (Cepheid, Sunnyvale, CA, USA) in a 120 clinical diagnostic laboratory. The sample was frozen for retrospective Nanopore sequencing.

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The sample was thawed and passed through a 0.45 µm filter prior to RNA extraction and 122 DNase treatment. cDNA was prepared and amplified using a Sequence-Independent-Single-

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Primer-Amplification method as described previously [11]. cDNA was used as input for a SQK-LS108 library preparation and sequencing on a R9.4.1 flow cell using a MinION device 125 (ONT).